Thrombin from Bovine Plasma

Product No. T7513
Storage Temperature: –20 °C
CAS RN 9002-04-4
EC 3.4.21.5
Synonym: Factor IIa

Product Description

Thrombin is an endolytic serine protease that selectively cleaves the Arg–Gly bonds of fibrinogen to form fibrin and release fibrinopeptides A and B.^1,2

The predominant form of thrombin in vivo is the zymogen, prothrombin (factor II), which is produced in the liver. The concentration of prothrombin in normal human plasma is 5–10 mg/dL.³ Prothrombin is a glycoprotein with a glycan content of ~12%.³

Prothrombin is cleaved in vivo by activated factor X, releasing the activation peptide and cleaving thrombin into light and heavy chains yielding catalytically active α-thrombin. α-Thrombin is composed of a light chain (A chain, MW ~6 kDA) and a heavy chain (B chain, MW ~31 kDa). These two chains are joined by one disulfide bond.⁴ The B chain of human thrombin consists of a peptide portion (MW 29,485 Da) and a carbohydrate portion (MW 2,334 Da) with N-linked glycosylation at three Asn residues.^5,6 Bovine thrombin contains 1.7% glucosamine, 1.8% sialic acid, 0.61% galactose, and 0.95% mannose.⁷

Thrombin also contains γ-carboxyglutamyl residues. These modified glutamyl residues are the result of carboxylation by a microsomal enzyme, vitamin K-dependent carboxylase. γ-Carboxyglutamyl residues are necessary for the Ca²⁺-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin.⁴ Prothrombin is activated in vivo on the surface of a phospholipid membrane that binds the amino terminus of prothrombin along with factors Va and Xa. The activation process starts slowly because factor V is activated to factor Va by the initial, small amount of thrombin.

Optimal cleavage sites for thrombin:²

1. A-B-Pro-Arg-||-X-Y where A and B are hydrophobic amino acids and X and Y are nonacidic amino acids
2. Gly-Arg-||-Gly

Thrombin from any mammalian species will clot the fibrinogen of any other mammalian species.⁸

Thrombin cleavage of fibrinogen occurs only at Arg residues; however, the cleavage site is not specific, resulting in 2 products. The primary cleavage product, fibrinopeptide A, is cleaved from fibrinogen after amino acid 16 and sometimes after amino acid 19, while a secondary cleavage product, fibrinopeptide B is produced by cleavage at amino acid 14.⁹

Thrombin does not require divalent metal ions or cofactors for activity. However, Na⁺-dependent allosteric activation of thrombin has been shown to play a role in defining the primary specificity of thrombin to cleave after Arg residues.¹⁰ Thrombomodulin serves as a cofactor for thrombin during the activation of protein C.¹¹

Under certain storage conditions, autolytic digestion of α-thrombin results in formation of β and γ-thrombins, which lack fibrinolytic activity, but retain some activity against synthetic peptide substrates and protein substrates other than fibrinogen.¹² This thrombin preparation is predominantly α-thrombin.

Thrombin (human and bovine) will catalyze the hydrolysis of several peptide p-nitroanilides, tosyl-Arg-nitrobenzyl ester, and thiobenzyl ester synthetic substrates.¹³

Catalytic pH range:¹⁴ 5–10, optimal pH:¹⁴ 8.3 thrombin precipitates ≤pH 5

Human isozymes pI range: 6.35–7.6.
Bovine pI range:¹⁵ 7.05–7.1

= 18.3 (human)¹⁶
= 19.5 (bovine)¹⁷

This product is lyophilized from a solution containing saline and sodium citrate buffer, pH 6.5.

Specific Activity: ≥2,000 NIH units/mg protein ( = 19.5)

Unit Definition: Activity is expressed in NIH units obtained by direct comparison to a NIH Thrombin Reference Standard, Lot K. The NIH assay procedure uses 0.2 mL of diluted plasma (1:1 with saline) as a substrate and 0.1 mL of albumin solution based on a modification of the method of Biggs.¹⁸ Only clotting times in the range of 15–25 seconds are used for determining thrombin activity. Optimal clotting temperature is 37 °C.

Thrombin concentrations in the literature are typically reported in terms of different units of activity.^18,19 Several conventions are used to express thrombin activity in the literature:

1 IOWA unit = 0.83 NIH unit
1 WHO unit = 1 NIH unit
1 NIH unit = 0.324 ± 0.073 mg
1 NIH unit = 1 USP unit

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

The product is soluble in water (10 mg/mL), yielding a clear solution.

Storage/Stability

Stock solutions can be prepared at a concentration of 100 units/mL in a 0.1% (w/v) BSA solution. Stock solutions remain active for one week at 0–5 °C. Solutions are most stable at pH 6.5, as a pH >7 will greatly reduce thrombin activity. Since thrombin solutions adsorb to glass, it is recommended to aliquot the solutions in plastic tubes and store at –20 °C for long-term storage.

Store the lyophilized powder at –20 °C.

Related Products

Synthetic Substrates:¹³

N-Benzoyl-Phe-Val-Arg-p-nitroanilide
N-Benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride
N-Benzoyl-Phe-Val-Arg 4-methoxy-β-naphthylamide
Boc-β-benzyl-Asp-Pro-Arg-7-amido-4-methylcoumarin
Boc-Val-Pro-Arg-7-amido-4-methylcoumarin
Sar-Pro-Arg p-nitroanilide
Thrombin generation chromogenic substrate
N-p-Tosyl-Gly-Pro-Arg 7-amido-4-methylcoumarin
N-(p-Tosyl)-Gly-Pro-Arg p-nitroanilide

Inhibitors:^20-22

Diisopropylfluorophosphate
Phenylmethylsulfonyl fluoride
AEBSF
Hirudin
Proflavine
Antithrombin III
α1-antitrypsin
α1-antiplasmin
Gabexate mesylate
Antipain
Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride